Technical Field
This disclosure relates to the targeting of genes to, and their integration into, an immunoglobulin heavy chain locus. The vectors, compositions, and methods disclosed herein are particularly useful for ex vivo accelerated antibody evolution.
Description of the Related Art
Monoclonal antibodies (mAbs) are well-established as therapeutics, diagnostics, and reagents for research, but their use is currently limited by the difficulties and costs associated with identifying mAbs with the required affinity and specificity for a desired target. Many targets of interest are highly conserved proteins, and mechanisms of immune regulation limit the variety of antibodies that can be obtained from a physiological immune response. In addition, many key therapeutic targets are cell surface proteins, which present particular challenges to mAb development because their physiologically active conformations are not readily recapitulated by purified proteins or membrane preparations used for immunization to elicit specific antibodies. These cell surface components include some especially high value targets for certain clinically useful contexts, such as cytokine receptors and G protein-coupled receptors.
Most current strategies for mAb discovery employ in vivo and/or in vitro approaches. In vivo approaches involve activation and selection of specific antibody-producing B cells by immunization, followed by generation of hybridomas (Kohler et al., 1975; Chiarella et al., 2008). This process is costly and time-consuming, since extensive screening and, in many cases, subsequent steps including affinity maturation are required to obtain mAbs with desired properties. It is also limited by immune tolerance, making antibodies that specifically recognize some antigens difficult or impossible to obtain. In addition, once a mAb has been identified there is not a straightforward path to further optimization of its affinity or functionality. In vitro approaches often rely on screening massive numbers of synthetic single-chain antibodies, typically displayed on phage (Winter et al., 1994; Bratkovic et al., 2010). These antibodies are expressed by cloned genes that encode linked immunoglobulin heavy chain variable (VH) and light chain variable (VL) regions derived from an immune repertoire, often from a convalescent individual (Grandea et al., 2010; Hammond et al., 2010). They can be further optimized by iterative PCR-based mutagenesis accompanied by selection in vitro, using high throughput approaches. However, success in the end depends on the quality of the starting libraries and their sources, and not all single-chain antibodies can be readily converted to natural antibodies for practical applications.
mAb discovery can also be carried out ex vivo in immortalized B cells. B cells display immunoglobulin (Ig) molecules on the cell surface, facilitating selection for antigen recognition. In some B cell lines, physiological pathways for immunoglobulin (Ig) gene diversification remain active, enabling evolution of high affinity antibodies in culture. The chicken B cell line, DT40, has proven especially adaptable for such purposes (Cumbers et al., 2002; Seo et al., 2005; Kajita et al., 2010). DT40 derives from a bursal lymphoma, and DT40 cells constitutively diversify their immunoglobulin heavy chain variable region (VH) and light chain variable region (VL) genes (Arakawa et al., 2004). Ongoing diversification occurs by two pathways, gene conversion and somatic hypermutation (Maizels et al., 2005). Briefly, most mutations are templated and arise as a result of gene conversion, with nonfunctional pseudo-V regions serving as donors for the transfer of sequences to the rearranged and transcribed V gene. A small fraction of mutations are nontemplated, and arise as a result of somatic hypermutation, the mutagenic pathway that generates point mutations in Ig genes of antigen-activated human and murine B cells. DT40 cells proliferate rapidly, with an 8-10 hr doubling time (compared to 20-24 hr for human B cell lines), and are robust to experimental manipulations including magnetic-activated cell sorting (MACS), fluorescence-activated cell sorting (FACS) and single-cell cloning. Most importantly, DT40 cells support very efficient homologous gene targeting (Buerstedde et al., 1991), so genomic regions can in many cases be replaced or modified as desired using appropriately designed homologous recombination strategies.
Despite the considerable potential of DT40 cells for antibody evolution, their utility has thus far been limited in practice because—as in other transformed B cell lines—Ig gene diversification occurs at less than 1% the physiological rate. Several approaches have been used to accelerate diversification in DT40 cells. This can be achieved by disabling the homologous recombination pathway (Cumbers et al., 2002), but cells thus engineered have lost the ability to carry out gene targeting, or to diversify their Ig genes by gene conversion, and diversification produces nontemplated point mutations, like those generated during antigen-driven somatic hypermutation in humans or mice. Diversification can also be accelerated by treatment of cells with the histone deacetylase inhibitor, trichostatin A (Seo et al., 2005). This approach increases the rate of gene conversion, but does not promote point mutagenesis, limiting potential diversity. Clearly there remains a need for more rapid and effective generation of coding sequence diversity in a target gene of interest such as an antibody-encoding gene. The presently described compositions and methods address this need and offer other related advantages.